The stress-responsive Hsp90 chaperone is required for the production of the genotoxin colibactin and the siderophore yersiniabactin by Escherichia coli

The genotoxin colibactin synthesized by Escherichia coli is a secondary metabolite belonging to the chemical family of hybrid polyketide/non-ribosomal peptide compounds. It is produced by a complex biosynthetic assembly line encoded by the pks pathogenicity island. The presence of this large cluster of genes in the E. coli genome is invariably associated with the High-Pathogenicity Island, encoding the siderophore yersiniabactin that belongs to the same chemical family as colibactin. The E. coli heat shock protein HtpG (Hsp90Ec) is the bacterial homolog of the eukaryotic molecular chaperone Hsp90 involved in the protection of cellular proteins against a variety of environmental stresses. In contrast to the eukaryotic Hsp90, the functions and client proteins of Hsp90Ec are poorly known. Here, we demonstrated that production of colibactin and yersiniabactin is abolished in the absence of Hsp90Ec We further characterized an interplay between the Hsp90Ec molecular chaperone and the ClpQ protease involved in colibactin and yersiniabactin synthesis. Finally, we demonstrated that Hsp90Ec is required for the full in vivo virulence of extraintestinal pathogenic E. coli This is the first report highlighting the role of heat shock protein Hps90Ec in the production of two secondary metabolites involved in E. coli virulence

Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage.

Colibactin is an assumed human gut bacterial genotoxin, whose biosynthesis is linked to the clb genomic island that has a widespread distribution in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumour formation and enhance the progression of colorectal cancer via cellular senescence and death induced by DNA double-strand breaks (DSBs); however, the chemical basis that contributes to the pathogenesis at the molecular level has not been fully characterized. Here, we report the discovery of colibactin-645, a macrocyclic colibactin metabolite that recapitulates the previously assumed genotoxicity and cytotoxicity. Colibactin-645 shows strong DNA DSB activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also delineate a complete biosynthetic model for colibactin-645, which highlights a unique fate of the aminomalonate-building monomer in forming the C-terminal 5-hydroxy-4-oxazolecarboxylic acid moiety through the activities of both the polyketide synthase ClbO and the amidase ClbL. This work thus provides a molecular basis for colibactin's DNA DSB activity and facilitates further mechanistic study of colibactin-related colorectal cancer incidence and prevention

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria

Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin

Colibactin represents an as-yet uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886 that uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block in its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal cancer-linked microbial chemical